Figure 4

Validation of isorhamnetin binding to Src kinase (a) Isorhamnetin docked in the ATP binding site of the human Src kinase domain. Relevant hydrogen bonds are shown in blue dashed lines. (b) Left panel: Chromatogram of dasatinib (10 mM, 5 μ L) on the K562/CMC system using 10 mM ammonium acetate buffer as the mobile phase. Mobile phases were added with (1) 0 μ mol/L; (2) 0.5 μ mol/L; (3) 1 μ mol/L ; (4) 2 μ mol/L; (5) 5 μ mol/L isorhamnetin, respectively. Right panel: Competitive displacement assay of isorhamnetin on K562/CMC columns (presented as k values) with different isorhamnetin concentrations (0, 0.5, 1, 2, and 5 μ M) added to the mobile phase. Protein levels of Src proteins in K562/CMC columns (C1, C2 and C3) and whole cells (W). (c) Selective ATP-competitive kinase assay of isorhamnetin through caliper mobility shift assay. (d) Representative example of isorhamnetin binding to Src kinase by SPR. Immobilized on Series S sensor chip CM5, and then the indicated concentrations of isorhamnetin were injected over the chip to obtain the sensorgrams. The 1:1 binding fitting model was used to determine the kinetic parameters. Error bars represent the standard deviation based on three independent samples (n = 3).